Identification of Regulators of the Rab GTPase Ypt11 Involved in Mitochondrial Inheritance

Event Center BC

Eukaryotic cells possess a variety of membrane-bound organelles that house specific biochemical functions essential to maintaining cellular health. Membrane fusion events and transport of entire organelles are regulated by molecular switch proteins called Rab GTPases. In yeast cells, the Rab GTPase Ypt11 (Rab11 in humans) coordinates inheritance of both mitochondria and Golgi membranes from mother cell to daughter cell. Ypt11 coordinates with a myosin, Myo2, to help transport Golgi and mitochondria along the actin cytoskeleton, through the bud neck, and into the growing bud. Previous studies have shown overexpression of Ypt11 in yeast results in hyperaccumulation of Golgi and mitochondrial membranes in the daughter cell—a phenotype that can be monitored using fluorescence microscopy. Cells turn off signaling by Rab GTPases using negative regulator proteins called Rab GAPs (GTPase Accelerating Proteins). It is unknown which Rab GAP is responsible for regulating Ypt11 activity at Golgi or mitochondria. Identifying a Rab GAP to counteract Ypt11 signaling would be an essential foundation in understanding regulation of organelle inheritance. We hypothesize that overexpression of a Rab GAP that counteracts Ypt11 signaling should restore normal localization of mitochondria and Golgi in strains overexpressing Ypt11. We generated high-copy plasmids to overexpress each of the nine known yeast Rab GAPs, monitoring mitochondrial localization using a yeast strain that includes a fluorescent protein tag on a mitochondrial marker protein. At the time of abstract preparation, a yeast strain appropriate for mitochondrial observation (OM45-mCherry) has been verified and experimental controls with overexpressed Ypt11 have been performed. Results of ongoing experiments examining the effects of over-expressed Rab GAPs on mitochondrial inheritance will be presented.